This invention relates to nucleic acid and amino acid sequences of a human cornichon protein and to the use of these sequences in the diagnosis, prevention, and treatment of developmental, reproductive, immunological, and neoplastic disorders.
Differentiation of tissues and determination of body plan in metazoans appears to be rooted in the synthesis of critical extracellular and intracellular proteins during oogenesis and embryogenesis. Determination of body plan is encrypted within embryonic cell lineages, and the fate of specific embryonic cell lineages is determined before fertilization, during oogenesis.
Oogenesis and embryogenesis are regulated by interactions between environmental, extracellular, and intracellular signals. Changes in signaling pathways caused by genetic mutation or biochemical modification can affect oogenesis and embryogenesis in a number of ways. Specifically, these changes may result in the failure of spermatozoa to fertilize the egg, in the premature death of the embryo, and in morphological changes during embryogenesis and during ontogeny.
Signaling pathways have been extensively studied during oogenesis and embryogenesis of the fruit fly, Drosophila melanogaster. Soon after fertilization, the Drosophila embryo has two axes of polarity, the anterior-posterior axis and the dorsal-ventral axis. These axes of polarity have been observed in all other metazoan embryos thus far studied. The shape of the Drosophila egg shows dorsal-ventral polarity at the time it is laid. Genetic studies have shown that three sequential signaling pathways establish the dorsal-ventral axis in the Drosophila embryo. The first of these signaling pathways takes place during oogenesis, when the germline-derived oocyte is surrounded by an epithelium of somatically-derived follicle cells. The follicle cells later secrete components of the eggshell. The oocyte produces a dorsalizing signaling ligand that is received by receptors on neighboring follicle cells and defines the polarity of both the embryo and the eggshell. The proposed ligand and receptor in this pathway are encoded by the genes gurken and torpedo, which are members of the transforming growth factor-xcex1 and the epidermal growth factor-receptor families, respectively. Spatial localization of the signal is achieved by localizing gurken mRNA to the dorsal anterior side of the oocyte. This is proximal to the asymmetrically positioned dorsal-anterior-localized oocyte nucleus (Morisato, D. and Anderson, K. V. (1995) Annu. Rev. Genet. 29:371-399).
A number of other genes are also required to ensure correct dorsalization of the oocyte. One of the eight which has been identified is cornichon (Morisato and Anderson (supra)). The predicted cornichon translation product is a 144 amino acid residue hydrophobic protein. Hydrophobic residues are clustered at three distinct regions: the N-terminus, the central region, and the C-terninus of the molecule. There are no putative transmembrane or signal sequences (Roth, S. et al. (1995) Cell 81:967-978). cornichon is thought to be involved in the membrane localization or proper activation of the gurken protein (Morisato and Anderson, supra).
Additional evidence suggests that cornichon is also absolutely required for the induction of the anterior-posterior axis. Mutations in cornichon prevent the formation of correctly polarized microtubule cytoskeleton. The microtubule cytoskeleton is required for proper localization of the anterior and posterior determinant genes bicoid and oskar and for the asymnmetric positioning of the oocyte nucleus (Roth et al., supra).
cornichon mRNA is expressed in the Drosophila ovary germarium, without a specific localization pattern. In early stages of oogenesis, it is present in the nurse cell oocyte cluster and is highly expressed in stage 1-6 egg chambers. Expression ceases during stage 7, but during stage 10, is reexpressed in the nurse cells (Roth, et al., supra).
The discovery of a new human cornichon protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of developmental, reproductive, immunological, and neoplastic disorders.
The invention features a substantially purified polypeptide, human cornichon protein (CORN), having the amino acid sequence shown in SEQ ID NO:1, or fragments thereof.
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof and a composition comprising said polynucleotide sequence. The invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:1, or fragments of said polynucleotide sequence. The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or variants of said polynucleotide sequence.
The invention also provides an isolated and purified sequence comprising SEQ ID NO:2 or variants thereof. In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2. The invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof.
The present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In yet another aspect, the expression vector containing the polynucleotide sequence is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding CORN under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified CORN having the amino acid sequence of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention also provides a purified antagonist of the polypeptide of SEQ ID NO:1. In one aspect the invention provides a purified antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:1.
Still further, the invention provides a purified agonist of the polypeptide of SEQ ID NO:1.
The invention also provides a method for treating or preventing a developmental disorder comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising purified CORN.
The invention also provides a method for treating or preventing a reproductive disorder comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising purified CORN.
The invention also provides a method for treating or preventing an immunological disorder comprising administering to a subject in need of such treatment an effective amount of an antagonist to CORN.
The invention also provides a method for treating or preventing a neoplastic disorder comprising administering to a subject in need of such treatment an effective amount of an antagonist to CORN.
The invention also provides a method for detecting a polynucleotide which encodes CORN in a biological sample comprising the steps of: a) hybridizing the complement of the polynucleotide sequence which encodes SEQ ID NO:1 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding CORN in the biological sample. In one aspect the nucleic acid material of the biological sample is amplified by the polymerase chain reaction prior to hybridization.